br Acknowledgements br Membrane bound proton
Membrane-bound proton-translocating inorganic pyrophosphatases (H-PPase; EC 18.104.22.168) belong to a recently identified category of proton pumps, distinct from F-, P-, and V-ATPases, which utilize pyrophosphate hydrolysis as the driving force for H movement across biological membranes . H-PPases have been characterized both at biochemical and genetic levels in various higher plants , , a few eubacteria , , , and more recently, in an archaea and some human pathogenic protozoa , , , , , , , , . In eukaryotes, H-PPase are associated to certain acidic compartments of the endomembrane system, namely, the vacuole and lysosomes of plant cells and the acidocalcisomes of trypanosomatids , . H-PPases have been reported to occur in the acidocalcisomal membranes of a number of pathogenic trypanosomatid and apicomplexan protozoa causative of endemic tropical diseases , , , and genes coding for these proteins have been cloned and sequenced in and . Moreover, data from genome project have revealed the presence of two different H-PPase genes in this human pathogen , , whereas sequences coming from unfinished microbial genome projects suggest that these proteins may be more widespread than previously thought . In any case, the information concerning the occurrence of H-PPase genes in non-photosynthetic protists is still rather scarce and unsystematic. H-PPases do not seem to be present in animals, thus, they might be potential targets for vaccines and drugs against pathogenic protists, most of them with an intracellular lifestyle. Consequently, studies on the genetics and c-met inhibitor of H-PPases in these organisms might be of great importance. We have performed a systematic search for genes coding for H-PPases in parasitic and free-living protists of different phylogenetic groups. Genomic DNA fragments of putative H-PPase genes were obtained by PCR using oligonucleotides designed from protein domains common to the well-known H-PPases of plants and the proteobacterium . The presence of the genes in the different genomes was further confirmed by Southern blot analyses. The sequences of the PCR-amplified DNA fragments allowed us to perform molecular phylogenetic studies on the H-PPases of protists and other organisms. The evolutionary and biomedical implications of these results are discussed. Materials and methods Organisms and growth conditions. Epimastigotes of T. cruzi Y strain were grown in filter-sterilized LIT medium with 10% heat-inactivated fetal calf serum (GIBCO), in tissue culture flasks at 28°C. Promastigotes of Leishmania major 252 strain were grown in M199 medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO). Other trypanosomatids (Leptomonas ctenocephali LV117 strain, Herpetomonas muscarum LRC-L130 strain, Phytomonas sp., Crithidia fasciculata LLM494 strain, and Endotrypanum schaudinni LLM865 strain) were grown in RPMI 1640 medium at 28°C. The photosynthetic heterokont Ochromonas danica SAG933-7 and the heterotrophic euglenoid Astasia longa 1204-17a were grown in SAG9 medium at 25°C. The hymenostomatid ciliates Tetrahymena pyriformis and Paramecium tetraurelia ATCC30759 were grown as previously described . Genomic DNA samples from the ciliates Histriculus cavicola (hipotrich) and Vorticella microstoma (peritrich), the slime mold Dictyostelium discoideum, and the apicomplexan parasites (P. falciparum, clinical sample, and T. gondii RH strain) were provided, respectively, by Prof. A. Torres (Universidad de Sevilla), Prof. J.J. Aragón (CSIC-UAM, Madrid), and the Servicio de Parasitologı́a, Instituto de Salud Carlos III, Madrid. Genomic DNA isolation. Protist cells were digested at 37°C for at least 1h with g/ml proteinase K in the presence of 10mM Tris–HCl, pH 8, 0.1M EDTA, 0.5% SDS, and g/ml DNAse-free RNAse. Samples were then subjected to extraction with phenol:chloroform followed by centrifugation. This procedure was repeated several times until no denatured proteins could be observed in the interphase. Total genomic DNA was eventually obtained by precipitation with 0.1 volumes of 3M sodium acetate (pH 5.5) and 2 volumes of ice-cold absolute ethanol. After washing with 70% ethanol, the final pellet was dried and resuspended in an appropriate volume of water.